PROJECT SUMMARY This project will test the hypothesis that bacterial bile salt hydrolase (BSH) activity regulates gut motility in a dose-dependent manner via signaling pathways in the enteric nervous system (ENS) that include RET, a tyrosine kinase receptor critical for ENS development and function, and TGR5, the only bile acid receptor known to be expressed by enteric neurons. To explore this hypothesis, I will integrate various technologies to produce functional readouts, combining a gnotobiotic mouse model of diet-dependent bile acid-mediated motility phenotypes with mass spectrometry, next-generation sequencing, and bioinformatics. In Aim 1, I propose to determine the extent to which gut motility is regulated by bacterial BSH activity. First, bacterial strains cultured from the microbiota of a single healthy Bangladeshi individual will be classified as possessing high, mid-level, or low BSH activity using an established in vitro screen. These strains have undergone preliminary screening to establish presence or absence of BSH; here I will characterize BSH activity with time-scale resolution down to 3 hours and with respect to the two predominant primary bile acid substrates in mice (taurocholic acid and tauro-beta-muricholic acid). Then, selected strains with high/mid/low BSH activity will be transplanted into gnotobiotic mice to determine (i) the extent to which BSH activity correlates with gut motility and (ii) whether BSH's motility effects are robust to taxonomic diversity (essential for understanding generalizability of the findings). In the future, I aim to initiate a clinical study to test the hypothesis that gut bacterial BSH activity correlates with motility in patients with diarrhea- or constipation-predominant irritable bowel syndrome, in order to identify subsets in whom bacterial bile acid metabolism could serve as a therapeutic target. In Aim 2, I propose to identify key ENS mediators of the effects of microbiome-encoded BSH activity on gut motility. To determine whether the TGR5 bile acid receptor is responsible for mediating this bile acid- dependent response, I will use conventionally raised and gnotobiotic Tgr5-/- mice. To identify key ENS molecular mediators and pathways, I will adopt TRAP-Seq (translating ribosome affinity purification sequencing), a technology developed to study distinct populations of the mouse brain, to profile the ENS in gnotobiotic mice. Using this approach, I will elucidate the effects of a cholekinetic agent (turmeric) and model human gut bacterial communities with defined BSH activity on the ENS transcriptome in the small intestine and colon, correlating these signals with measured transit times and bile acid profiles. Together, these data obtained from humanized gnotobiotic mice will help dissect the extent to which bacterial bile acid metabolism regulates gut motility and provide mechanistic insights regarding interactions between dietary ingredients, the gut microbiome, and ENS signaling pathways that regulate motility. Follow-up future studies will involve transplanting intact uncultured microbiota from selected well-phenotyped humans with motility disorders into gnotobiotic mice.